Antimycobacterial activity of some Brazilian
indigenous medicinal drinks
Oliveira, D.G.1,2; Prince, K.A.2; Higuchi, C.T.2; Santos, A.C.B.2; Lopes, L.M.X.3; Simões, M.J.S.2; Leite, C.Q.F.2*
1Departamento de Análises Clinicas, Faculdade de Farmácia Bioquímica, Universidade do Oeste Paulista, UNOESTE, Presidente Prudente, SP, Brasil. 2Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, UNESP, Araraquara, SP, Brasil.
3Departamento de Quimica Organica, Instituto de Química, Universidade Estadual Paulista, UNESP, Araraquara, SP, Brasil.
*Autor Correspondente: Clarice Queico Fujimura Leite
-Departamento de Ciências Biológicas - Faculdade de Ciências Farmacêuticas - Universidade Estadual Paulista, UNESP - Rodovia Araraquara - Jaú, km 01 - CEP: 14801-902 - Araraquara - SP, Brasil - Telefone: (16) 3301-6953 - e-mail: leitecqf@fcfar.unesp.br
Recebido 20/08/07 / Aceito 10/12/07
Farmacêuticas Básica e Aplicada
Journal of Basic and Applied Pharmaceutical Sciences Rev. Ciênc. Farm. Básica Apl., v. 28, n.2, p.165-169, 2007ISSN 1808-4532
ABSTRACT
Tuberculosis (TB) is a very serious problem worldwide and the increasing number of multiple drugs resistant TB cases makes the search for new anti-TB drugs an urgent need. Indigenous knowledge about the use of native plants to treat illnesses has contributed to the discovery of new medicines. In this study, the antimycobacterial activity of seven medicinal drinks was assessed: Ananas sativus (hydroalcoholic fruit extract), Aristolochia triangularis (aqueous and hydroalcoholic leaf, root and stem extracts),
Bromelia antiacantha (hydroalcoholic fruit extract), Stryphnodendron adstringens (hydroalcoholic bark
extract), Tabebuia ovellanedae (hydroalcoholic bark extract), Vernonia polyanthes (hydroalcoholic root extract), all used by the Vanuíre indigenous community in the treatment of respiratory diseases. The activity was evaluated by using a time-to-kill assay, in which
Mycobacterium tuberculosis H37Rv was cultured on
Löwenstein-Jensen medium, after thirty minutes, one, three, six, twelve and twenty-four hours contact of the bacteria with each drink. Within half to one hour contact, the hydroalcoholic drinks of A. triangularis, S. adstringens,
T. ovellanedae and V. polyanthes reduced the bacterial
growth by 2 orders of magnitude in CFU/mL, and all bacterial growth was absent after three hours contact. In contrast, no mycobactericidal effect was detected in the aqueous extract of A. triangularis or in the hydroalcoholic beverages of A. sativus and B. antiacantha, even after twenty-four hours contact.
Keywords: Tuberculosis; indigenous drinks; Aristolochia triangularis; Stryphnodendron adstringens; Tabebuia ovellanedae; Vernonia polyanthes.
INTRODUCTION
Mycobacterium tuberculosis is responsible for more
human deaths than any other single microbial species. Although there are anti-tubercular agents that are reasonably
effective in treating tuberculosis (TB), the misuse of these agents has led to an increasing prevalence of multiple-drug resistant strains (Vieira et al., 2007). Therefore, it is necessary to prioritize the research on new drugs with activity against
M. tuberculosis. The relation between the risk of developing
TB and social-economic factors is widely known (Portal da Saúde, 2007) and the problem of TB directly affects the low-income indigenous communities (Basta et al., 2004). However, during a period of ten years (1994 to 2004) of an epidemiological survey carried out among 208 inhabitants of the Vanuíre indigenous community established in Arco-Íris, São Paulo State, Brazil, only one case of tuberculosis was notified. The medicinal plants, in the form of crude extracts, infusions or beverages, are widely used by this community to treat common infections.
In this context, we decided to evaluate the antimycobacterial activity of beverages used by this community. After analyzing the responses to a questionnaire presented to members of the Vanuíre indigenous community, six plants and seven beverages were selected: Ananas sativus (boiled fruit extract); Vernonia polyanthes (roots hydroalcoholic extract); Stryphnodendron adstringens (bark hydroalcoholic extract); Tabebuia ovellanedae (wood or bark hydroalcoholic extract), Bromelia antiacantha (boiled fruit extract), Aristolochia triangularis (leaf, root and stem hydroalcoholic and aqueous extract). These beverages are used by this community for the treatment of respiratory diseases. They were tested against Mycobacterium
tuberculosis H37Rv.
MATERIAL AND METHODS
Ethical aspects
Plant materials
Fresh plant material (at least 500g) from each species selected (Table 1) was harvested in the western region of São Paulo State, near Arco Íris, SP, Brazil. The exact location is indicated in Table 1. The plant specimens were collected with the aid of the local indigenous community and exsiccates were prepared, classified and deposited at the University of West São Paulo (UNOESTE), Presidente Prudente, SP. Collected plants were used to prepare the medicinal drinks according to the popular methods. The sugarcane juice spirit "cachaça" (with 30% alcohol), used to prepare the hydroalcoholic formulations, was acquired from the local community.
Microorganism
Mycobacterium tuberculosis H37Rv - ATCC 27294
was used to evaluate the antimycobacterial activity of the beverages. The bacterium was stored on Lowenstein Jensen (DIFCO) medium slants until use.
Antimycobacterial kinetic assay
Time-killing curves (in triplicate assay) were obtained with serial, decimal dilutions (10-1 to 10-4) of M. tuberculosis prepared from 106 CFU/mL culture in Middlebrook 7H9 broth (BBL): 1mL of the 10-1 and 10-2 bacterial dilutions were mixed into 9mL of the beverages, giving final bacterial concentrations of 10-3 and 10-4,
Scientific name Popular name Collecting site Part of plant Extract
Ananas sativus Schultz Abacaxizinho Tupi Paulista/SP Fruit hydroalcoholic
Aristholochia triangularis Cham Cipó mil homem Paranapanema/SP Leaves, Roots
and Stem
aqueous
Aristholochia triangularis Cham Cipó mil homem Paranapanema/SP Leaves, Roots
and Stem
hydroalcoholic
Bromelia antiacantha Bertol Gravatá do Mato Anaurilândia/MS and
Teodoro Sampaio/SP
Fruit hydroalcoholic
Stryphnodondron adstringens
Mart
Barbatimão Paraguaçu Paulista/SP
Bark hydroalcoholic
Tabebuia ovellanedae Lor. Ex
Griseb
Ipê-roxo P. Prudente/SP Bark hydroalcoholic
Vernonia polyanthes Less Assa peixe Paranapanema/SP Root hydroalcoholic
Table 1 - Details of plant species and medicinal drinks.
respectively. The mixture was kept shaking for contact periods of half, three, six, twelve and twenty-four hours, after which 0.1 mL of each beverage sample was inoculated on Löwestein - Jensen (L-J) medium (DIFCO) slants and incubated at 35-37ºC for 21 days, under weekly observation. To monitor the viability of the bacteria, 0.1mL of the 10-3, 10-4 and 10-5 dilutions were also cultured on the L-J medium and incubated in the same way. The influence of the alcohol present in the beverages was also evaluated by determining the antimycobacterial activity of the sugarcane spirit used. Isoniazid (Sigma) was used as a reference drug to assay bacterial sensitivity .
RESULTS
On the basis of the questionnaire presented to members of the Vanuíre indigenous community, six plant species and seven formulations used to treat pulmonary diseases by this community were selected (Table 1). During this survey it was noted that mainly the older people of the community made use of these formulations. When the herbs were not used directly to cure infections, they were used to help in other pulmonary treatments.
Antimycobacterial activity results are presented in Table 2. The beverages prepared with A. triangularis (aqueous), A. sativus and B. antiacantha did not show any antimycobacterial activity. The number of viable bacteria remained similar to the control even after 24h mycobacteria/ beverage contact. However, the hydroalcoholic beverages
prepared from A. triangularis, T. ovellanedae and V.
polyanthes showed promising activity against M. tuberculosis, even after discounting the poisonous effect of
the alcohol against viable cells of M. tuberculosis (see 'cachaça' in Table 2). The hydroalcoholic beverages of A.
triangularis and T. ovellanedae showed reductions of two
logarithmic orders of magnitude in the number of viable bacteria after thirty minutes mycobacteria/beverage contact (reduction of 100-fold in viability) and absence of viable mycobacteria after one hour contact. For hydroalcoholic beverages of S. adstringens and , 100-fold reduction was obtained in 1hour and, after three hours contact, no mycobacterial growth at all was observed.
DISCUSSION
Plants have provided many medicinal drugs in the past and remain a potential source of therapeutic agents to this day (Phillipson, 2003). The use of plants with medicinal action has grown, despite all the advances made in medicine, even in developed countries, due to several factors such as the confidence of the populations that use them, their ease of acquisition, and their low cost (Récio et al., 1989). Public scepticism about the ability of allopathic medicines to be free from adverse effects, or to cure chronic conditions, have contributed to consumer demand for high quality herbal medicinal products (Phillipson, 2003). Hence, indigenous plants are used worldwide as medicines, particularly in the developing countries.
A growing number of chemical and pharmacological studies is proving the effectiveness and the existence of
Beverages Extraction Colony-forming Units (CFU)/mL
½ hour (log)1 hour (log) 3 hour (log) 6 hour (log) 12 hour (log)
A sativus hydroalcoholic 4 4 3.9 4 4.1
A.triangularis aqueous 4.2 4.1 4 4 4
A.triangularis hydroalcoholic 2 absent absent absent absent
B.antiacanta hydroalcoholic 3.9 4 3.9 4.1 4.1
S.adstringns hydroalcoholic 3.1 2.5 absent absent absent
T.ovellanedae hydroalcoholic 1.9 absent absent absent absent
V. polyanthes hydroalcoholic 2.9 2 absent absent absent
Cachaça 30% alcohol 3.5 3 2 1.2 0.8
Table 2 - Results of mycobacterial activity of beverages in CFU/mL a, b
medicinal properties in a great variety of plants. Antimycobacterial activity has been found in several species of plants and some classes of natural compound such as the terpenoids (Cantrell et al., 2001) and physalines (Pietro et al., 2000; Okunade et al., 2004) were indicated as responsible for the biological activity against M. tuberculosis.
In this study, we selected six plant species and seven formulations used by the Vanuíre indigenous community to treat pulmonary diseases. This community, consisting of 208 inhabitants, is established in Arco-Íris district, São Paulo State, Brazil. The community is composed of people belonging to four indigenous nations (Krenak, Kerena, Kaiwa and Kaigang), which come from several Brazilian regions.
Antimycobacterial activity was confirmed in the hydroalcoholic beverages from A. triangularis, S.
adstringens, T. ovellanedae and V. polyanthes.
There are several species of Aristolochia that exhibit similar properties, and they are also used as antiseptic, antitussive, expectorant, antiasthmatic medicines, and to treat lung inflammation (Lopes & Humpfer 1997). A. triangularis, collected in the Brazilian states of Rio Grande do Sul (114) and Paraná (Lopes & Humpfer 1997), showed a significant presence of kaurene diterpenes. In an extensive review of natural products with anti-mycobacterial activity, Coop (2003) registered the valuable antitubercular action of diterpenes. Other secondary metabolites, such as the tetrandine (Lopes & Humpfer 1997) isolated from species of Aristolochia, also proved to have a strong antibiotic effect against M. tuberculosis. The leaves and roots of V. polyanthes have been used in traditional medicine, as a diuretic, balsamic and antirheumatic (Lorenzi, 2000). The effect of the crude extract of V. polyanthes on blood pressure and renal excretion
Antimycobacterial activity of indigenous drinks
a control: log
10 (CFU/mL)= 4.2
was studied by Romanezi et al. (2003).
Owing to its high concentration of tannin, the bark of S. adstringens is used in home-made medicine against hemorrhage, diarrhea, hemorrhoids, wounds and conjunctivitis (Lorenzi, 2002).
The phytochemical analyses of Tabebuia species revealed the presence of naphthoquinones, mainly lapachol, that shows antitumoral activity and antimicrobial activity (Lorenzi, 2002). The antibacterial activity of T. impetiginosa against Helicobacter pylori was reported by Park et al. (2006). Nevertheless, we could find no research about the antimycobacterial activity of Tabebuia ovellanedae.
In spite of the wide use of the V. polyanthes, T.
ovellanedae, S. adstringens and A. triangularis species by
the Vanuíre community, the activity against M. tuberculosis of these plants, especially in the beverages, has still not been reported. Another relevant result emerging in this research is that, when the aqueous and hydroalcoholic beverages of A. triangularis were compared, it was observed that only the hydroalcoholic extract was active against M. tuberculosis, indicating the lesser polar character of the active constituents. This can be explained by the high hydrophobicity of the mycobacterial cell wall (Rando et al., 2002).
It is notable that, according to Basta et al. (2004), the incidence of tuberculosis among the Surui Indians (Rondônia) was as high as 2519 per 100.000 inhabitants. This reinforces the idea that tuberculosis is an infect-contagious and social disease and that the indigenous community is very susceptible to tuberculosis. However, the low incidence of tuberculosis (only one case) in the Vanuíre community during ten years surveillance may be explained by their use of beverages with antitubercular activity.
RESUMO
Atividade antimicobacteriana de algumas bebidas medicinais indígenas
A tuberculose é um problema mundial sério e vem aumentando significativamente com o surgimento de novos casos de multi-drogas resistência necessitando-se pesquisar novas drogas. Os conhecimentos indígenas sobre o uso de plantas nativas para a cura de certas doenças têm contribuído para a obtenção de novos medicamentos. No presente estudo, foi avaliada a atividade anti-micobacteriana de sete bebidas medicinais indígenas: Ananas sativus (extrato hidro-alcoólico de fruta), Aristolochia triangularis (extrato aquoso e hidro-alcoólico de folhas, raízes desidratadas), Bromelia
antiacantha (extrato hidro-alcoólico de fruta), Stryphnodendron adstringens (extrato hidro-alcoólico de
casca), Tabebuia ovellanedae (extrato hidro-alcoólico de casca), Vernonia polyanthes (extrato hidro-alcoólico de raiz) usados pela comunidade indígena Vanuíre situada em Arco-Íris, Estado de São Paulo no tratamento de
afecções respiratórias. A atividade antituberculose foi avaliada pela exposição do Mycobacterium tuberculosis H37Rv as beberagens por trinta minutos, um, três, seis, doze e vinte e quatro horas. Nos tempos de exposição de trinta minutos a uma hora as bebidas hidro-alcoólicas de A. triangularis, S. adstringens, T. ovellanedae e V.
polyanthes, reduziram em 2log10 a multiplicação bacilar
(determinada em UFC/mL). A ausência de crescimento bacteriano foi verificado a partir da terceira hora de exposição da bactéria a bebida. Por outro lado, nenhuma atividade micobactericida foi verificada nas soluções aquosas de A. triangularis e nas bebidas hidro-alcoólicas de A. sativusand, B. antiacantha mesmo após 24 horas de exposição.
Palavras-chave: Tuberculose; beberagens indígenas; Aristolochia triangularis; Stryphnodendron adstringens; Tabebuia ovellanedae; Vernonia polyanthes.
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